High-voltage activated Ca channels (CaV1/2 channels) convey calcium influx that drives a vast array of biological functions, including cardiac excitation, neurotransmission, and memory formation. As such, they are tightly regulated by calcium-dependent (CDI) and voltage dependent inactivation (VDI). While the initial steps of these processes are well-known, little is known about the subsequent steps. These unknowns are prominent gaps in the field, given that defects in such inactivation cause various neuronal and cardiac calcium channelopathies. The overall goal of this proposal is to identify the intermediate events that follow depolarization or Ca binding to CaM and to elucidate the final conformations of CDI and VDI through 3 aims: (1) Does Calmodulin(CalVI) induce CDI by moving among different channel domains? Preliminary data indicate that mutations in an upstream EF-hand domain on C-terminus of channel could dramatically reduce CDI. We have also identified potential alternate CaM binding sites computationally. We will (a) identify the structural and functional roles of the EF-hand motif critical in determining CDI, (b) explore whether CaM leaves its well-known IQ binding site, and (c) monitor resulting conformation changes of intracellular domains using TIRF/FRET imaging under patch clamp. (2) Does mechanical torsion on intracellular loops induce Ca2+ channel inactivation? If mechanical coupling of the S6 gates with the intracellular loops on the channel play a role in inactivation, we should be able to alter channel inactivation by physically constraining these loops. We will inducibly invoke such constraints by rapamycin-induced heterodimerization between FKBP-channel loops and membrane-localized Lyn-FRB domains. (3) What are the ultimate end-stage processes of CDI and VDI? Three major models for end-stage mechanisms of CDI and VDI are: (i) hinged-lid occlusion, (ii) pore-collapse, or (ill) allosteric inhibition of opening. Preliminary data suggests that CDI occurs through allosteric modulation. The relevant molecular machinery for pore collapse is possibly conserved in Ca channels. We will distinguish among these mechanisms by: (a) co-expressing l-ll loop peptide which could act as an excess of free 'lids'that speed inactivation, (b) undertaking an alanine scan of selectivity filter regions to test for pore collapse, and (c) using FRET to image possible pore-collapse associated with channel inactivation. Relevance: Through these experiments we would fill a large void in our present understanding of Ca channel inactivation and, more generally, ion channel regulation. It would also provide a stepping stone to advance our understanding of the mechanistic consequences of lethal channelopathies raising hope for targeted therapeutic interventions.